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Method for producing artificial bear gall powder through engineering bacteria fermentation

A technology of engineering bacteria and bear bile powder, applied in the biological field, can solve the problems of a large number of organic solvents, cumbersome steps, and unfavorable environmental protection

Pending Publication Date: 2017-02-01
SHANGHAI KAIBAO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are also reports on the production of its active ingredient TUDCA by chemical synthesis, but also because the steps are cumbersome, and the production process needs to use a large amount of organic solvents, which is not conducive to environmental protection

Method used

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  • Method for producing artificial bear gall powder through engineering bacteria fermentation
  • Method for producing artificial bear gall powder through engineering bacteria fermentation
  • Method for producing artificial bear gall powder through engineering bacteria fermentation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Whole-gene synthesis of codon-optimized 7α-HSDH and 7β-HSDH genes

[0071] 7α-HSDH and 7β-HSDH are 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase, respectively;

[0072]The two 7α-HSDHs were derived from: Clostridium sardiniense strain DSM599, GenBank accession number: JN191345.1) and Escherichia coli strain TW14359, GenBank accession number: CU928163.2),

[0073] The two 7β-HSDH genes were derived from: Clostridium sardiniense strain DSM599, GenBank accession number: JN191345.1) and Ruminococcus gnavus strain N53, GenBank accession number: KF052988.1.

[0074] Using http: / / www.jcat.de / online software, Escherichia coli as host bacteria, codon optimization of the whole gene sequence according to the codon preference, and whole gene synthesis, respectively recorded as α1, α2, β1 , β2.

[0075] The codon-optimized 7α-HSDH derived from Clostridium sardiniense (denoted as α 1 ) gene sequence as shown in SEQ ID No 1; codon-optimized 7α-HSDH derived fr...

Embodiment 2

[0077] Construction of engineered bacteria

[0078] 1) Expanded culture using pMD as a carrier with a synthetic target gene α 1 、α 2 , β 1 , β 2 coli, and DH5α-pETM6 strain: take 10μl sample, add 10ml of LB (Amp + ) medium, cultivated on a shaker at 37° C. for 12 to 16 hours, and the shaker speed was 225 rpm.

[0079] 2) Use the plasmid extraction kit purchased from Shanghai Chuangying Biotechnology Co., Ltd. to extract the plasmids of the above bacteria, and operate according to the operation instructions of the kit.

[0080] 3) For the extracted pMD-α 1 , pMD-α 2 , pMD-β 1 , pMD-β 2 The plasmid and the expression vector pETM6 were subjected to double enzyme digestion, and the enzyme digestion system was as follows:

[0081]

[0082] Digest at 37°C for 2 to 4 hours, and purify the target fragment with a gel recovery kit.

[0083] 4) The recovered 850bp target gene fragment α 1 、α 2 , β 1 , β 2 , respectively, and the 5.6kb pETM6 carrier fragment, the connecti...

Embodiment 3

[0108] Cultivation and fermentation of engineered bacteria

[0109] 1) Apply the engineering bacteria stored in the -80°C refrigerator to the LB solid plate medium containing 100 mg / L ampicillin by streaking method, and activate and culture at 37°C for 12 hours;

[0110] The composition of the LB solid medium is as follows: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, agarose 15.0g / L.

[0111] 2) Pick a single colony from the LB solid plate medium, inoculate it in the LB liquid medium supplemented with 100mg / L ampicillin, and culture it with shaking at 225rpm and 37°C for 12 hours;

[0112] The composition of described LB liquid medium is as follows: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L;

[0113]3) The above culture was inoculated into liquid 1XM9 medium at a ratio of 1:50, cultured with shaking at 225rpm / min and 37°C for 5 hours, and 1mM isopropyl-β-D-thiogalactopyranoside ( IPTG), adjusted to 30 ° C for 3 hours;

[0114] 4) prepare the N...

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Abstract

The invention relates to a method for producing and preparing artificial bear gall powder through the optimized engineering bacteria fermentation technology, and belongs to the field of biotechnology. Escherichia coli simultaneously expressing 7alpha-hydroxysteroid dehydrogenase (7alpha-HSDH) and 7beta-hydroxysteroid dehydrogenase (7beta-HSDH) is used for liquid fermentation, taurochenodeoxycholic acid in poultry bile products like chicken gall powder is directly converted into tauroursodeoxycholic acid in a certain proportion, the fermentation process is simple and quick, conditions are mild, and pollution is not caused. Fermentation results are stable and controllable, the conversion rate is high, and the number of intermediate products is small. The method plays an important role in the biotechnology production of bear gall powder substitute resources.

Description

technical field [0001] The invention relates to a method for producing artificial bear bile powder by optimized fermentation of engineering bacteria, in particular to a method for converting taurochenodeoxycholic acid (TCDCA) in poultry bile products through genetically modified non-pathogenic microorganisms. ) components in situ into a certain proportion of tauroursodeoxycholic acid (TUDCA) method, belongs to the field of biotechnology. Background technique [0002] Bear bile powder is a dry product obtained from the bile drainage of the black bear Selenaretos thibetanus Cuvier after gallbladder surgery. Clinically, it is often used for heat convulsions, epilepsy, convulsions, and jaundice in children; external use is used to treat carbuncles, hemorrhoids, red eyes, cloudy eyes, etc. Bear bile powder mainly contains bile acid, and its main active ingredient is tauroursodeoxycholic acid (TUDCA). The Chinese Pharmacopoeia stipulates that the content of TUDCA should not be le...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P41/00C12P33/00C12N15/70C12R1/19C12R1/145C12R1/01
CPCC12P33/00C12P41/00
Inventor 赵淑娟王峥涛史杰杨莉周吉燕胡之璧张金家
Owner SHANGHAI KAIBAO PHARMA
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